All vertebrate haemoglobins (Hb) are tetramers consisting of two alpha (.alpha.) globin chains and two beta (.beta.) globin sub-units each carrying an iron-containing prosthetic group, haem. The oxygen binding to the four sub-units within the tetramer is co-operative (ie binding of oxygen at one haem group facilitates the binding of oxygen at the other haem groups on the same globin tetramer and conversely, the unloading of oxygen at one haem unit facilitates the unloading of oxygen at the others).
During vertebrate evolution the amino acid sequence of globins has diverged considerably, and the sequence identitv between Hbs from the most distantly-related vertebrate species, (eg human and fish), is only about 50%. That between human and crocodile haemaglobins, for example, is about 60%. Despite this sequence variation, vertebrate Hbs have generally retained their tertiary and quaternary structures (Camardella et al., 1992 J. Mol. Biol. 224, 449-460) and the key residues necessary for haem-haem interaction.
The oxygen affinity of human Hb is modulated by various metabolites, such as 2,3-diphosphoglycerate (DPG) and H.sup.+. They facilitate unloading of oxygen to actively respiring tissues. This general phenomenon is called the heterotropic allosteric effect.
In particular, the oxygen affinity of Hb is affected by the concentration of CO.sub.2, such that high concentrations of CO.sub.2, (with a partial pressure, "PCO.sub.2 " of about 40 mm Hg, as will be found in actively respiring tissues) cause a reduction in the oxygen affinity, thereby facilitating the unloading of oxygen to the tissues. This phenomenon is known as the CO.sub.2 effect. In human Hb, CO.sub.2 binds directly to the .alpha.-amino groups of the .alpha. and .beta. globin submits to form carbamino groups and reduces the oxygen affinity by stabilising the low affinity (T) quaternary structure (Kilmartin & Rossi-Bernardi, 1969 Nature 222, 1243-1246). The CO.sub.2 effect is one of the principal heterotropic allosteric effects.
Vertebrate species have adapted to strikingly diverse environmental conditions, and the haemoglobin molecule has evolved to meet a wide range of respiratory needs in such environments. For example, the oxygen affinity of crocodile Hb is marketly reduced by physiological concentrations of CO.sub.2 (40 torr). Bauer et al. (1981, J. Biol. Chem. 256, 8429-8435) showed that the large reduction in the oxygen affinity of crocodile Hb is not caused by the binding of CO.sub.2 to the .alpha. amino groups as in human Hb. Carbon dioxide (CO.sub.2) dissolves in aqueous solutions (such as blood) to give HCO.sub.3 --. The marked reduction in the oxygen affinity in crocodile Hb is caused by the binding of bicarbonate ions and this phenomenon is called the "bicarbonate effect". A much larger proportion of Hb-bound oxygen can be released and utilised in the tissues and this enables crocodiles to stay under water for a long time. It has been postulated (Perutz et al., 1981 Natue 291, 682-684) that this effect is due to bicarbonate ions binding to residues Lys 82 and Glu 144 of the .beta. globin molecule.
There is interest in developing "artificial blood" products. Such products would eliminate the chance of infection due to a patient receiving infected blood from a donor, and generally simplify the blood transfusion process. Clearly, in order to perform all the functions of natural blood, the artificial product must be capable of transporting oxygen.
Unfortunately, haemoglobin outside the environment of a red blood cell (RBC) has too high an affinity to release much oxygen to the tissues. This is because RBCs contain large amounts of DPG which, as mentioned above, is one of the metabolites which lowers the oxygen affinity of Hb.
Consequently, there is a need for a stable, artificial blood product with improved oxygen affinity characteristics which will deliver oxygen more effectively to respiring tissues.